US11046945B2 - Labeled glutaminase proteins, isolated glutaminase protein mutants, methods of use, and kit - Google Patents

Labeled glutaminase proteins, isolated glutaminase protein mutants, methods of use, and kit Download PDF

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US11046945B2
US11046945B2 US14/771,085 US201414771085A US11046945B2 US 11046945 B2 US11046945 B2 US 11046945B2 US 201414771085 A US201414771085 A US 201414771085A US 11046945 B2 US11046945 B2 US 11046945B2
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gac
leu
gly
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gls
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Clint A. STALNECKER
Jon W. Erickson
Sekar RAMACHANDRAN
Rick Cerione
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Cornell University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
    • C12Y305/01002—Glutaminase (3.5.1.2)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/978—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • G01N2333/98—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/20—Screening for compounds of potential therapeutic value cell-free systems

Definitions

  • the present invention relates to labeled glutaminase proteins, isolated glutaminase protein mutants, methods of screening for compounds that allosterically bind to glutaminase proteins, methods of identifying compounds that inhibit or stabilize tetramer formation of glutaminase proteins, and screening kits for compounds that inhibit or stabilize tetramer formation of glutaminase.
  • GLS glutaminase
  • glutamine addiction The elevation in glutamine metabolism exhibited by cancer cells (“glutamine addiction”) is thought to be critical for sustaining their proliferative capacity as well as for other aspects of their transformed phenotypes (Wise et al., “Glutamine Addiction: A New Therapeutic Target in Cancer,” Trends Biochem. Sci.
  • GAC GLS splice variant
  • DON for Diazo-O-norleucine
  • glutamine derivative that forms a stable acyl-enzyme intermediate with the catalytic serine residue responsible for deamidase activity.
  • BPTES bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide
  • a more recently identified class of allosteric inhibitors of GAC which offer the advantage of being highly specific in their ability to inhibit the growth and invasive activity of cancer cells, while having little effect on normal (non-transformed) cells, is represented by the benzophenanthridinone, designated as 968 (Wang et al., “Targeting Mitochondrial Glutaminase Activity Inhibits Oncogenic Transformation,” Cancer Cell 18:207-219 (2010); Katt et al., “Dibenzophenanthridinones as Inhibitors of Glutaminase C and Cancer Cell Proliferation,” Mol. Cancer Ther. 11:1269-1278 (2012)).
  • the specificity exhibited by 968 for inhibiting the transformed features of cancer cells holds exciting promise for selectively attacking those metabolic changes critical for malignant transformation. However, thus far very little is known regarding how 968 binds to GAC and the mechanisms by which it blocks GAC activation.
  • the present invention is directed to overcoming these and other deficiencies in the art.
  • One aspect of the present invention relates to a labeled glutaminase (GLS) protein comprising a GLS protein and a fluorescent reporter group attached to the GLS protein, where the fluorescent reporter group is attached to the GLS protein within the glutaminase domain pfam04960 of GLS.
  • GLS labeled glutaminase
  • Another aspect of the present invention relates to an isolated glutaminase (GLS) protein or protein fragment comprising a mutated glutaminase domain pfam04960 of SEQ ID NO:19.
  • GLS glutaminase
  • a further aspect of the present invention relates to a method of screening for compounds that allosterically bind to a glutaminase (GLS) protein.
  • This method involves providing the labeled GLS protein of the present invention under conditions effective for the fluorescent reporter group attached to the GLS protein to produce fluorescence at a first level.
  • the labeled GLS protein is contacted with one or more candidate compounds.
  • Candidate compounds where said contacting causes the fluorescent reporter group to emit fluorescence at a level above or below the first level are identified as being compounds capable of allosteric binding to the GLS protein.
  • Yet another aspect of the present invention relates to a method of identifying compounds that inhibit or stabilize tetramer formation of glutaminase (GLS) protein.
  • This method involves providing a first labeled GLS dimer protein comprising a GLS protein and a fluorescent donor attached to the GLS dimer protein.
  • a second labeled GLS dimer protein comprising a GLS protein and a fluorescent acceptor attached to the GLS protein is also provided, where binding of the first labeled GLS protein and the second labeled GLS protein forms a GLS protein tetramer and results in an interaction between the fluorescent donor and the fluorescent acceptor which produces a fluorescence resonance energy transfer at a first level.
  • the first labeled GLS dimer protein and the second labeled GLS dimer protein are contacted under conditions effective for the first labeled GLS dimer protein and the second labeled GLS dimer protein to bind and form a GLS protein tetramer.
  • the GLS protein tetramer is contacted with a candidate compound.
  • the method further involves detecting whether said contacting with the candidate compound alters the fluorescence resonance energy transfer at the first level. Detection of the fluorescence resonance energy transfer at the first level indicates that the candidate compound neither inhibits nor stabilizes GLS protein tetramer formation and detection of the fluorescence resonance energy transfer at a level above or below the first level indicates that the candidate compound inhibits or stabilizes tetramer formation of GLS protein.
  • kits for compounds that inhibit or stabilize tetramer formation.
  • the kit includes a first labeled GLS dimer protein comprising a GLS protein and a fluorescent donor attached to the GLS dimer protein.
  • a second labeled GLS dimer protein comprising a GLS protein and a fluorescent acceptor attached to the GLS protein. Binding of the first labeled GLS protein and the second labeled GLS protein forms a GLS protein tetramer and results in an interaction between the fluorescent donor and the fluorescent acceptor which produces a fluorescence resonance energy transfer.
  • the binding of 968 to a mutant form of a GLS protein splice variant that is trapped in the monomeric state is characterized, and it is shown that this binding correlates with inhibition of recombinant GLS in a real-time coupled binding and activity assay.
  • Novel fluorescence read-outs are used that, for the first time, allow definitive demonstration that 968 and related compounds directly bind to GLS.
  • the binding of 968 to the GLS splice variant correlates well with its inhibition of the protein's activity, and importantly, with its ability to block the growth of transformed cells.
  • FIGS. 1A-F illustrate the development of a fluorescence assay to monitor subunit interactions.
  • FIG. 1A is a schematic model of a FRET assay developed to detect dynamic tetramer formation.
  • FIG. 1B illustrates the kinetics of labeling wild type (“WT”) GAC with ALEXA FLUOR® 488 succinimidyl ester analyzed by SDS-PAGE and visualized under UV illumination. 488-labeled GAC was analyzed using analytical gel filtration to illustrate the purification of a covalently modified GAC along with eluting at the same volume as an equivalent concentration of unlabeled WT-GAC.
  • WT wild type
  • ALEXA FLUOR® 488 succinimidyl ester analyzed by SDS-PAGE and visualized under UV illumination.
  • 488-labeled GAC was analyzed using analytical gel filtration to illustrate the purification of a covalently modified GAC along with eluting at the same volume as an equivalent concentration of unlabele
  • FIG. 1C shows that 25 nM 488-WT-GAC fluorescence is quenched upon addition of QSY® 9-WT-GAC (acceptor) in a dose dependent manner (quantified in FIG. 1D ), and can be rescued by addition of a 10-fold excess unlabeled WT-GAC.
  • FRET values from FIG. 1C were overlaid with concentration dependent activation (in absence of P i ) of WT-GAC (right axis) in the presence of 20 mM glutamine measured in an independent two-step activity assay.
  • FIG. 1E QSY® 9-GAC-488-GAC tetramers were equilibrated by adding 100 nM QSY® 9-WT-GAC to 25 nM 488-WT-GAC, and the effects of phosphate addition on FRET was followed by addition of various phosphate concentrations at 630 seconds.
  • FIG. 1F FRET values that resulted from injection of phosphate from FIG. 1E (left axis) were overlaid with phosphate activation of 50 nM WT-GAC in the presence of 20 mM glutamine (right axis) measured in an independent two-step activity assay.
  • FIGS. 2A-B illustrate that mutating specific residues at the GAC monomer and dimer interface traps mutants in a defined oligomeric state.
  • FIG. 2A is the crystal structure of the tetramer form of GAC (PDB 3SS3), highlighting critical contacts for monomer-monomer contact (top) and dimer-dimer contact (bottom). Interfaces are presented as B-factor representations and not cartoons to facilitate visualization of the interactions.
  • FIG. 2B is an overlay of Superdex200 preparative chromatograms of purified mutants.
  • FIGS. 3A-C illustrate that WT GAC accesses monomer, dimer, and larger oligomeric species in a concentration and phosphate dependent manner.
  • FIG. 3A is a graph of analytical gel filtration profiles of WT GAC from a 250 ⁇ L injection of either 5 mg/mL or 0.5 mg/mL samples in the presence or absence of 50 mM K 2 HPO 4 in the gel filtration buffer showing a strong correlation of oligomeric state with GAC concentration and inorganic phosphate, whereas the same conditions of the D391K-GAC ( FIG. 3B ) or K316E-D391K-R459E-GAC ( FIG. 3C ) does not affect oligomerization. Notably, D391K-GAC ( FIG. 3B ) was found to have two populations when 0.5 mg/mL samples were injected but not 5 mg/mL, characteristic of a monomer and dimer population that is concentration dependent.
  • FIGS. 4A-F define the oligomeric species of GAC mutants.
  • multi-angle light scattering profiles of WT-GAC FIG. 4A
  • D391K-GAC FIG. 4B
  • K316E-D391K-R459E-GAC FIG. 4C
  • K316E-GAC FIG. 4D
  • SEC and subsequent MALS analysis where elution of each species was monitored using refractive index (left axis).
  • refractive index left axis
  • light scattering data was collected and then used to calculate the molecular weight and polydispersity for the species eluted (right axis).
  • Reference lines for the molecular weights of the monomer, dimer, and tetramer forms of GAC are included at 58 kD, 116 kD, and 230 kD respectively.
  • 200 nM of QSY® 9-WT-GAC, QSY® 9-D391K-GAC, or QSY® 9-K316E-D391K-R459E was added to 20 nM of 488-WT-GAC.
  • WT GAC and GAC mutants were titrated and added to an assay of 20 mM glutamine in the absence of phosphate to show no concentration dependent activation was observed of purified GAC mutants.
  • FIGS. 5A-C illustrate that the effects of allosteric inhibitors BPTES and 968 on tetramer formation leads to direct binding read out of 968 and 488-GAC.
  • FIG. 5A is a graph showing that addition of 10 ⁇ M BPTES to an equilibrated sample of 20 nM 488-GAC and 200 nM QSY® 9-GAC induces tetramer formation that is not reversible by addition of a 10-fold excess of unlabeled GAC, whereas addition of 25 ⁇ M of 968 induces a marked quench in 488-GAC fluorescence with partial recovery by the addition of a 10-fold excess of unlabeled GAC.
  • FIG. 5A is a graph showing that addition of 10 ⁇ M BPTES to an equilibrated sample of 20 nM 488-GAC and 200 nM QSY® 9-GAC induces tetramer formation that is not reversible by addition of a 10-fold excess of un
  • FIG. 5B fluorescence quenching upon addition of 968 to 10 nM 488-GAC in the absence of QSY® 9-GAC shows a concentration dependent quenching interaction.
  • FIG. 5C is an overlay of 968 inhibition of 10 nM WT-GAC activity and 968 quenching of 10 nM 488-GAC fluorescence.
  • FIGS. 6A-F illustrate coupling real time drug binding with enzymatic activity.
  • FIG. 6A is a schematic model of a real time drug binding assay coupled to a real-time activity assay. Binding is first monitored by observing 488-GAC fluorescence, followed by observation of NADH fluorescence that is produced upon the addition of the substrate for GAC, glutamine, and the activator inorganic phosphate, in the presence of 10 Units/mL glutamate dehydrogenase (GDH) and 2 mM NAD + .
  • GDH glutamate dehydrogenase
  • FIGS. 6C-D illustrate the results of a coupled real time binding and activity assay of 10 nM 488-GAC and 10 nM WT-GAC using 968 and a less potent 968-analogue, WPK968.
  • FIGS. 6E-F illustrate the results of a coupled real time binding and activity assay of 10 nM 488-GAC with 968-analogues 031 and 742, previously reported as GAC inhibitors.
  • FIGS. 7A-B illustrate that the small molecule 968 preferentially binds to GAC monomer.
  • FIG. 7A is a plot illustrating 488 fluorescence quenching of 20 nM 488-labeled WT GAC, dimer, and monomer GAC mutants upon 968 titration.
  • FIG. 7B shows in vitro inhibition curves of 50 nM (closed circles) and 5 nM WT-GAC (open circles) with increasing concentrations of pre-incubated 968, where primary GAC species at each concentration is a dimer/tetramer or monomer/dimer, respectively.
  • FIGS. 8A-C illustrate the identification of a small molecule probe labeling site.
  • 0.6 mg/mL 488 labeled KGA and GAC samples were incubated with 25 ⁇ g/mL porcine trypsin (Sigma) on ice for 15 minutes or 60 minutes, at which point Soy Bean Trypsin Inhibitor (SBTI, Sigma) was added to make 20 ⁇ g/mL.
  • SBTI Soy Bean Trypsin Inhibitor
  • Loading buffer was added and samples were heated at 95° C. for 2 min and ran on a precast 4-12% Tris-Glycine gel (Invitrogen) for SDS PAGE.
  • the gel was visualized under UV illumination and then transferred to a PVDF membrane to be developed following Western immunoblot with rabbit HRP conjugated anti-GAC antibody raised against the C-terminal GAC peptide (SEQ ID NO:3) highlighted in FIG. 8C .
  • the anti-GAC antibody recognition sequence (SEQ ID NO:13) is set forth in bold in FIG. 8C .
  • SEQ ID NO: 3 is the full sequence set forth in FIG. 8C
  • SEQ ID NO: 13 is residues 531-550 of the sequence set forth in FIG. 8C .
  • FIG. 8B the same protocol was followed as in FIG.
  • FIGS. 9A-B illustrate that the alternate splice variant KGA behaves like GAC in a FRET assay.
  • FIGS. 10A-B illustrate measuring of the monomer-monomer binding affinity.
  • An appropriate volume of 4.4 ⁇ M QSY® 9-D391K-GAC was added to 5 nM 488-D391K-GAC and 520 nm emission was monitored for 10 minutes.
  • FRET values from FIG. 10A were plotted in a sigma plot and fit to non-linear regression simple ligand binding equation (line).
  • FIG. 11 is a sequence alignment of four mutated GLS proteins according to one aspect of the present invention, including mouse GAC (SEQ ID NO:12), human GAC (SEQ ID NO:11), mouse KGA (SEQ ID NO:10), and human KGA (SEQ ID NO:9).
  • the present invention relates to labeled glutaminase proteins and isolated glutaminase protein mutants.
  • the present invention relates to methods of using these proteins in a method for screening for compounds that allosterically bind to a glutaminase protein and a method of identifying compounds that inhibit or stabilize tetramer formation of a glutaminase protein.
  • the present invention further relates to a screening kit for compounds that inhibit or stabilize tetramer formation.
  • the present invention relates to a labeled glutaminase (GLS) protein comprising a GLS protein and a fluorescent reporter group attached to the GLS protein, where the fluorescent reporter group is attached to the GLS protein within the glutaminase domain pfam04960 of GLS.
  • GLS labeled glutaminase
  • glutaminase proteins include wild type proteins, including, for example, GLS isoforms GAC and KGA from human and mouse.
  • the GLS isoforms GAC and KGA are splice variants of each other. Specifically, their C-terminal regions are unique (i.e., residues 550-603 of mouse GAC and residues 550-674 of mouse KGA).
  • human GAC and KGA proteins each have unique C-terminal regions (i.e., residues 545-598 of human GAC and residues 545-669 of human KGA).
  • amino acid residues 1-72 comprise the mitochondrial targeting sequence.
  • the human GAC protein is set forth in GenBank Accession No. NP_001243239.1, which is hereby incorporated by reference in its entirety, and has the amino acid sequence of SEQ ID NO:1, as follows:
  • the cDNA sequence encoding the human KGA protein, infra is set forth in GenBank Accession No. NM_014905.4, which is hereby incorporated by reference in its entirety, and has the nucleotide sequence of SEQ ID NO:2, as follows:
  • the mouse GAC protein is set forth in GenBank Accession No. NP_001106854.1, which is hereby incorporated by reference in its entirety, and has the amino acid sequence of SEQ ID NO:3, as follows:
  • the cDNA sequence encoding the above mouse GAC protein is set forth in GenBank Accession No. NM_001113383.1, which is hereby incorporated by reference in its entirety, and has the nucleotide sequence of SEQ ID NO:4, as follows:
  • the human KGA protein is set forth in GenBank Accession No. NP_055720.3, which is hereby incorporated by reference in its entirety, and has the amino acid sequence of SEQ ID NO:5, as follows:
  • GenBank Accession No. NM_001256310.1 which is hereby incorporated by reference in its entirety, and has the nucleotide sequence of SEQ ID NO:6, as follows:
  • the mouse KGA protein is set forth in GenBank Accession No. NP_001074550.1, which is hereby incorporated by reference in its entirety, and has the amino acid sequence of SEQ ID NO:7, as follows:
  • the cDNA sequence encoding the above mouse KGA protein is set forth in GenBank Accession No. NM_001081081.2, which is hereby incorporated by reference in its entirety, and has the nucleotide sequence of SEQ ID NO:8, as follows:
  • GLS proteins are also contemplated as labeled glutaminase proteins according to this aspect of the present invention.
  • Other GLS proteins include GLS proteins from other animal sources, i.e., GAC and KGA proteins from non-mouse and non-human sources. According to one embodiment, these and other GLS proteins have an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, and/or SEQ ID NO:7.
  • Percent identity refers to the comparison of one amino acid (or nucleic acid) sequence to another, as scored by matching amino acids (or nucleic acids). Percent identity is determined by comparing a statistically significant number of the amino acids (or nucleic acids) from two sequences and scoring a match when the same two amino acids (or nucleic acids) are present at a position. The percent identity can be calculated by any of a variety of alignment algorithms known and used by persons of ordinary skill in the art.
  • GLS proteins according to this embodiment of the present invention may be isolated from a sample or tissue by methods commonly used by persons of ordinary skill in the art, or produced recombinantly, e.g., from a GLS encoding nucleic acid molecule.
  • cDNA sequences that encode GLS proteins are set forth above and include, without limitation, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8.
  • Expression of a GLS protein can be carried out by introducing a nucleic acid molecule encoding the GLS protein into an expression system of choice using conventional recombinant technology. Generally, this involves inserting the nucleic acid molecule into an expression system to which the molecule is heterologous (i.e., not normally present). The introduction of a particular foreign or native gene into a mammalian host is facilitated by first introducing the gene sequence into a suitable nucleic acid vector.
  • Vector is used herein to mean any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc., which is capable of replication when associated with the proper control elements and which is capable of transferring gene sequences between cells.
  • the term includes cloning and expression vectors, as well as viral vectors.
  • the heterologous nucleic acid molecule is inserted into the expression system or vector in proper sense (5′ ⁇ 3′) orientation and correct reading frame.
  • the vector contains the necessary elements for the transcription and translation of the inserted GLS protein coding sequence.
  • Recombinant genes may also be introduced into viruses, including vaccinia virus, adenovirus, and retroviruses, including lentivirus.
  • Recombinant viruses can be generated by transfection of plasmids into cells infected with virus.
  • Suitable vectors include, but are not limited to, the following viral vectors such as lambda vector system gt11, gt WES.tB, Charon 4, and plasmid vectors such as pBR322, pBR325, pACYC177, pACYC184, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37, pKC101, SV 40, pBluescript II SK+/ ⁇ or KS+/ ⁇ (see “Stratagene Cloning Systems” Catalog (1993) from Stratagene, La Jolla, Calif., which is hereby incorporated by reference in its entirety), pQE, pIH821, pGEX, pFastBac series (Invitrogen), pET series (see F.
  • viral vectors such as lambda vector system gt11, gt WES.tB, Charon 4, and plasmid vectors such as
  • Recombinant molecules can be introduced into cells via transformation, particularly transduction, conjugation, mobilization, or electroporation.
  • the DNA sequences are cloned into the vector using standard cloning procedures in the art, as described by Sambrook et al., Molecular Cloning: A Laboratory Manual , Cold Springs Laboratory, Cold Springs Harbor, N.Y. (1989), which is hereby incorporated by reference in its entirety.
  • host-vector systems may be utilized to express the GLS protein-encoding sequence in a cell.
  • the vector system must be compatible with the host cell used.
  • Host-vector systems include but are not limited to the following: bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA; microorganisms such as yeast containing yeast vectors; mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus); and plant cells infected by bacteria.
  • the expression elements of these vectors vary in their strength and specificities. Depending upon the host-vector system utilized, any one of a number of suitable transcription and translation elements can be used.
  • mRNA messenger RNA
  • telomere a DNA sequence that directs the binding of RNA polymerase and thereby promotes mRNA synthesis.
  • the DNA sequences of eukaryotic promoters differ from those of prokaryotic promoters.
  • eukaryotic promoters and accompanying genetic signals may not be recognized in or may not function in a prokaryotic system, and, further, prokaryotic promoters are not recognized and do not function in eukaryotic cells.
  • SD Shine-Dalgarno
  • Promoters vary in their “strength” (i.e., their ability to promote transcription). For the purposes of expressing a cloned gene, it is desirable to use strong promoters in order to obtain a high level of transcription and, hence, expression of the gene. Depending upon the host cell system utilized, any one of a number of suitable promoters may be used. For instance, when cloning in E.
  • coli its bacteriophages, or plasmids, promoters such as the PH promoter, T7 phage promoter, lac promoter, trp promoter, recA promoter, ribosomal RNA promoter, the P R and P L promoters of coliphage lambda and others including, but not limited to, lacUV5, ompF, bla, lpp, and the like, may be used to direct high levels of transcription of adjacent DNA segments. Additionally, a hybrid trp-lacUV5 (tac) promoter or other E. coli promoters produced by recombinant DNA or other synthetic DNA techniques may be used to provide for transcription of the inserted gene.
  • promoters such as the PH promoter, T7 phage promoter, lac promoter, trp promoter, recA promoter, ribosomal RNA promoter, the P R and P L promoters of coliphage lambda and others including, but not limited to, lacUV5, omp
  • Bacterial host cell strains and expression vectors may be chosen which inhibit the action of the promoter unless specifically induced.
  • the addition of specific inducers is necessary for efficient transcription of the inserted DNA.
  • the lac operon is induced by the addition of lactose or IPTG (isopropylthio-beta-D-galactoside).
  • IPTG isopropylthio-beta-D-galactoside.
  • Specific initiation signals are also required for efficient gene transcription and translation in prokaryotic cells. These transcription and translation initiation signals may vary in “strength” as measured by the quantity of gene specific messenger RNA and protein synthesized, respectively.
  • the DNA expression vector which contains a promoter, may also contain any combination of various “strong” transcription and/or translation initiation signals. For instance, efficient translation in E. coli requires a Shine-Dalgarno sequence about 7-9 bases 5′ to the initiation codon (ATG) to provide a ribosome binding site. Thus, any SD-ATG combination that can be utilized by host cell ribosomes may be employed.
  • Such combinations include but are not limited to the SD-ATG combination from the cro gene or the N gene of coliphage lambda, or from the E. coli tryptophan E, D, C, B or A genes. Additionally, any SD-ATG combination produced by recombinant DNA or other techniques involving incorporation of synthetic nucleotides may be used.
  • any number of suitable transcription and/or translation elements including constitutive, inducible, and repressible promoters, as well as minimal 5′ promoter elements may be used.
  • the GLS protein-encoding nucleic acid, a promoter molecule of choice, a suitable 3′ regulatory region, and if desired, a reporter gene are incorporated into a vector-expression system of choice to prepare a nucleic acid construct using standard cloning procedures known in the art, such as described by Sambrook et al., Molecular Cloning: A Laboratory Manual , Third Edition, Cold Spring Harbor: Cold Spring Harbor Laboratory Press, New York (2001), which is hereby incorporated by reference in its entirety.
  • the nucleic acid molecule encoding a GLS protein is inserted into a vector in the sense (i.e., 5′ ⁇ 3′) direction, such that the open reading frame is properly oriented for the expression of the encoded GLS protein under the control of a promoter of choice.
  • Single or multiple nucleic acids may be ligated into an appropriate vector in this way, under the control of a suitable promoter, to prepare a nucleic acid construct.
  • the isolated nucleic acid molecule encoding the GLS protein has been inserted into an expression vector, it is ready to be incorporated into a host cell.
  • Recombinant molecules can be introduced into cells via transformation, particularly transduction, conjugation, lipofection, protoplast fusion, mobilization, particle bombardment, or electroporation.
  • the DNA sequences are incorporated into the host cell using standard cloning procedures known in the art, as described by Sambrook et al., Molecular Cloning: A Laboratory Manual , Second Edition, Cold Springs Laboratory, Cold Springs Harbor, N.Y. (1989), which is hereby incorporated by reference in its entirety.
  • Suitable hosts include, but are not limited to, bacteria, virus, yeast, fungi, mammalian cells, insect cells, plant cells, and the like.
  • an antibiotic or other compound useful for selective growth of the transformed cells is added as a supplement to the media.
  • the compound to be used will be dictated by the selectable marker element present in the plasmid with which the host cell was transformed. Suitable genes are those which confer resistance to gentamycin, G418, hygromycin, puromycin, streptomycin, spectinomycin, tetracycline, chloramphenicol, and the like.
  • reporter genes which encode enzymes providing for production of an identifiable compound, or other markers which indicate relevant information regarding the outcome of gene delivery, are suitable. For example, various luminescent or phosphorescent reporter genes are also appropriate, such that the presence of the heterologous gene may be ascertained visually.
  • the GLS protein of the labeled GLS protein according to this aspect of the present invention is not a wild type protein but is mutant protein.
  • the GLS protein may be a human or mouse GAC or KGA protein as set forth above in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7, except that the protein has, for example, one or more amino acid substitutions, or one or more deletions or insertions.
  • such a GLS protein mutant has an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, and/or SEQ ID NO:7, and has, for example, one or more amino acid substitutions, or one or more deletions or insertions.
  • the GLS protein is a mutant protein having an amino acid sequence comprising SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12 (set forth infra), or a protein that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and/or SEQ ID NO:12.
  • the GLS protein is a mutant protein having an amino acid sequence comprising three amino acid substitutions corresponding to K316E/D391K/R459E of mouse GAC protein (i.e., SEQ ID NO:3).
  • the mutated GLS protein is a GLS monomer that is incapable of forming a GLS dimer.
  • the mutation(s) stabilizes the GLS protein in a monomer form and prevents the mutated GLS protein from forming a dimer.
  • the GLS protein is a GLS protein or GLS protein fragment comprising the glutaminase domain pfam04960 of GLS as discussed infra.
  • the GLS protein is a protein or protein fragment comprising the pfam04960 domain of SEQ ID NO:18, as follows:
  • the labeled GLS protein of the present invention has a fluorescent reporter group attached to the GLS protein within the glutaminase domain pfam04960 of GLS.
  • the glutaminase domain pfam04960 of GLS includes amino acid residues 73-550 of mouse GAC (SEQ ID NO:3) and mouse KGA (SEQ ID NO:7), and amino acid residues 73-545 of human GAC (SEQ ID NO:1) and human KGA (SEQ ID NO:5).
  • the glutaminase domain pfam04960 of GLS is the consensus sequence of SEQ ID NO:18, set forth supra.
  • the fluorescent reporter group is, according to one embodiment, attached within amino acid residues 244-530 of human GAC protein (SEQ ID NO:1) and human KGA protein (SEQ ID NO:5), or within amino acid residues 249-535 of mouse GAC protein (SEQ ID NO:3) and mouse KGA protein (SEQ ID NO:7).
  • Suitable fluorescent reporter groups for carrying out this and other aspects of the present invention include a wide variety of fluorescent probes commonly used and widely available on the market. These fluorescent reporter groups could be any synthetic fluorophores that are either sensitive to their local environment, such as exhibiting a change in fluorescence in response to changes in immediate polarity, or sufficient reporter groups that produce fluorescence resonance energy transfer (FRET) between a donor fluorescent probe and an acceptor absorbant probe. Fluorescent reporter groups that are environamentally-sensitive can exhibit a change in fluorescence intensity, fluorescence life-time, or changes in their excitation or emission profiles.
  • FRET fluorescence resonance energy transfer
  • Environmentally sensitive fluorophores suitable for use in the present invention include, but are not limited to, derivatives of 7-aminocoumarin, fluorescein, rhodamine, pyrene, naphthalenes, dansyl chloride (5-dimethylaminonaphthalene-1-sulfonyl chloride), pyridyloxazole, dapoxyl, and nitrobenzoxadiazole (NBD).
  • probes include, but are not limited to, fluorescent dyes from MOLECULAR PROBES® (Thermo Fisher Scientific, Inc.), such as the ALEXA FLUOR® series, DyLight FLUOR® series, rhodamine and/or fluorescein derivatives, Coumarin, Pacific GreenTM, Oregon Green®, Cy® 3, Pacific OrangeTM, Texas Red®, and Cy® 5.
  • fluorescent dyes from MOLECULAR PROBES® (Thermo Fisher Scientific, Inc.), such as the ALEXA FLUOR® series, DyLight FLUOR® series, rhodamine and/or fluorescein derivatives, Coumarin, Pacific GreenTM, Oregon Green®, Cy® 3, Pacific OrangeTM, Texas Red®, and Cy® 5.
  • These probes are attached to a GLS protein through direct covalent interaction with a native or mutated amino acid sidechain having a terminal amino or thiol reactive group (i.e., lysine and cysteine).
  • the modification is performed by combining the GLS protein and the reporter group containing a reactive side-group together under conditions that allow reaction of the side-group attached to the reporter group with the GLS protein.
  • the groups used to covalently attach reporter groups to amino or thiol wellding amino acids are widely available, and typically have a reactive side-group attached to the reporter group of choice that has a known reaction with amino or thiol groups.
  • these groups can include, but are not limited to, isothiocyanates, succinimydyl esters, sulfotetrafluorophenyl (STP) esters, tetrafluorophenol (TFP) esters, sulfodichlorophenol (SDP) esters, carbonyl azides, and sulfonyl chlorides.
  • STP sulfotetrafluorophenyl
  • TFP tetrafluorophenol
  • SDP sulfodichlorophenol
  • carbonyl azides and sulfonyl chlorides.
  • thiol group modification these groups include, but are not limited to, iodoacetamides, maleimides, 6-bromoacetyl-2-dimethylaminonaphthalene (badan), and acrylodan.
  • These reactive groups can react with either native amino acids, or amino acids that have been inserted through molecular genetic approaches at a defined position.
  • the fluorescent reporter group is covalently attached to the GLS protein.
  • attachment of the fluorescent reporter group to the GLS protein is carried out by covalent modification of a native amino group presented by a lysine amino acid by a succinimidyl ester derivative of ALEXA FLUOR® 488 or QSY® 9 to form a stable amide-linked adduct comprising the reporter group and amino acid side chain.
  • Another aspect of the present invention relates to an isolated glutaminase (GLS) protein or protein fragment comprising a mutated glutaminase domain pfam04960 of SEQ ID NO:19, as follows:
  • Specific isolated GLS protein mutants comprising a mutated glutaminase domain pfam04960 of SEQ ID NO:19 include, for example and without limitation, an amino acid sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12 or a protein or protein fragment that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and/or SEQ ID NO:12.
  • SEQ ID NO:9 mutated human KGA
  • SEQ ID NO:10 mutated mouse KGA
  • SEQ ID NO:11 mutated human GAC
  • SEQ ID NO:12 mutated mouse GAC
  • FIG. 11 An alignment of SEQ ID NO:9 (mutated human KGA), SEQ ID NO:10 (mutated mouse KGA), SEQ ID NO:11 (mutated human GAC), and SEQ ID NO:12 (mutated mouse GAC) is set forth in FIG. 11 .
  • These isolated glutaminase protein mutants differ from SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7, by a three amino acid substitution.
  • the mutated mouse KGA differs from SEQ ID NO:7 by the three amino acid substitution K316E/D391K/R459E.
  • the mutated human KGA differs from SEQ ID NO:5 by the three amino acid substitution K311E/D386K/R454E.
  • the mutated mouse GAC differs from SEQ ID NO:3 by the three amino acid substitution K316E/D391K/R459E.
  • the mutated human GAC differs from SEQ ID NO:1 by the three amino acid substitution K311E/D386K/R454E.
  • nucleic acid coding sequence can encode for any one of the mutated GLS proteins of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12. Specifically, for every codon there are usually at least three different variations of possible nucleotide sequences.
  • Non-limiting examples of cDNA coding for the mutated GLS proteins of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12 are set forth as follows.
  • SEQ ID NO:14 One exemplary cDNA coding for the mutated human KGA of SEQ ID NO:9 is SEQ ID NO:14, as follows:
  • SEQ ID NO:15 One exemplary cDNA coding for the mutated human GAC of SEQ ID NO:11 is SEQ ID NO:15, as follows:
  • SEQ ID NO:16 One exemplary cDNA coding for the mutated mouse KGA of SEQ ID NO:10 is SEQ ID NO:16, as follows:
  • SEQ ID NO:17 One exemplary cDNA coding for the mutated mouse GAC of SEQ ID NO:12 is SEQ ID NO:17, as follows:
  • a further aspect of the present invention relates to a method of screening for compounds that allosterically bind to a glutaminase (GLS) protein.
  • This method involves providing the labeled GLS protein of the present invention under conditions effective for the fluorescent reporter group attached to the GLS protein to produce fluorescence at a first level.
  • the labeled GLS protein is contacted with a candidate compound.
  • Candidate compounds where said contacting causes the fluorescent reporter group to emit fluorescence at a level above or below the first level are identified as being compounds capable of allosteric binding to the GLS protein.
  • GLS protein In carrying out this and other aspects of the present invention, providing the labeled GLS protein can be accomplished as described supra. In addition, the GLS protein used in this and other aspects of the present invention is as described supra.
  • This method of the present invention may be carried out in a cell, but is not necessarily carried out in a cell.
  • the GLS protein When carried out in a cell, the GLS protein may be recombinantly expressed, as described supra, and the fluorescent reporter is attached to the GLS protein as described supra to provide the labeled GLS protein.
  • the labeled GLS protein by its fluorescent label, emits fluorescence at first level (e.g., a particular wavelength or intensity associated with the fluorescent reporter group).
  • a candidate compound is a compound that causes the fluorescent reporter group to emit a fluorescence at a level above or below the first level, or causes a detectable change in fluorescence (e.g., a shift in the fluorescence wavelength or intensity, or a change in fluorescence lifetime) of the fluorescent reporter group.
  • Detecting a change in fluorescence in this and other aspects of the present invention may be carried out by visual observation. Alternatively, detecting a change in fluorescence may be carried out with a spectrophotometer, or a microscope or macroscope system coupled to a camera or photomultiplier tube. Coupled with proper instrumentation, the optical readout can be followed in real time to obtain spatio-temporal information (functional intracellular imaging).
  • the GLS protein is, according to one embodiment, a monomer. According to an alternative embodiment, the GLS protein is a dimer.
  • FIG. 6A One embodiment of this method of the present invention is illustrated in FIG. 6A .
  • a GLS protein dimer is shown to be labeled with ALEXA FLUOR® 488 succinimidyl ester (“488-GAC”). This GLS protein dimer emits a high fluorescence. When contacted with candidate compound 968, 488-GAC emits a low fluorescence.
  • candidate compound 968 binds the GLS protein and causes the fluorescent reporter group ALEXA FLUOR® 488 attached to the GLS protein to emit a fluorescence at a level below the first level (i.e., the level of fluorescence emitted by 488-GAC in the absence of contact with candidate compound 968).
  • the method according to this aspect of the present invention may further involve contacting the GLS protein, after identifying candidate compounds, with glutamine under conditions effective to activate the GLS protein.
  • NADH is detected following the contacting with the GLS protein, after said identifying with glutamine.
  • Candidate compounds (1) where NADH is detected are identified as being compounds that do not inhibit GLS protein activity and (2) where NADH is not detected are identified as being compounds that do inhibit GLS protein activity. This embodiment is also illustrated in FIG.
  • the labeled GLS protein bound by a candidate compound is contacted with glutamine (Gln+P i ) under conditions to activate the GLS protein to form a GLS protein tetramer (illustrated in the right side of the schematic in FIG. 6A ).
  • the tetramer form of the GLS protein catalyzes the reaction of glutamine to NADH, as illustrated in FIG. 6A .
  • the detection of NADH in carrying out this method of the present invention is indicative of the candidate compound not inhibiting GLS protein activity (despite binding GLS protein).
  • the candidate compound is identified as a GLS protein activity inhibitor.
  • Yet another aspect of the present invention relates to a method of identifying compounds that inhibit or stabilize tetramer formation of glutaminase (GLS) protein.
  • This method involves providing a first labeled GLS dimer protein comprising a GLS protein and a fluorescent donor attached to the GLS dimer protein.
  • a second labeled GLS dimer protein comprising a GLS protein and a fluorescent acceptor attached to the GLS protein is also provided, where binding of the first labeled GLS protein and the second labeled GLS protein forms a GLS protein tetramer and results in an interaction between the fluorescent donor and the fluorescent acceptor which produces a fluorescence resonance energy transfer at a first level.
  • the first labeled GLS dimer protein and the second labeled GLS dimer protein are contacted under conditions effective for the first labeled GLS dimer protein and the second labeled GLS dimer protein to bind and form a GLS protein tetramer.
  • the GLS protein tetramer is contacted with a candidate compound.
  • the method further involves detecting whether said contacting with the candidate compound alters the fluorescence resonance energy transfer at the first level. Detection of the fluorescence resonance energy transfer at the first level indicates that the candidate compound neither inhibits nor stabilizes GLS protein tetramer formation and detection of the fluorescence resonance energy transfer at a level above or below the first level indicates that the candidate compound inhibits or stabilizes tetramer formation of GLS protein.
  • the first and second GLS proteins are wild type proteins.
  • the first and second proteins are GLS isoforms selected from GAC and KGA.
  • the first and second proteins are a single GLS isoform, e.g., the first and second proteins are both GAC or the first and second proteins are both KGA.
  • the GLS dimer proteins may be labeled with labels discussed supra.
  • the labels are capable of forming FRET pairs, where fluorescence energy from a fluorescent donor probe can be transferred to an absorbant but not necessarily fluorescent accepter probe (e.g., non-fluorescent QSY dyes available from MOLECULAR PROBES® (Thermo Fisher Scientific, Inc.)). Any FRET pair is suitable for this method of the present invention involving the readout of inhibition or stabilization of GLS protein tetramer formation.
  • the fluorescent donor is ALEXA FLUOR® 488 succinimidyl ester and the fluorescent acceptor is QSY® 9 succinimidyl ester, both of which are MOLECULAR PROBES® obtainable from Thermo Fisher Scientific, Inc.
  • Other donors and acceptors are well known and can also be used.
  • a first labeled GLS dimer protein comprising a GLS protein and a fluorescent donor attached to the GLS dimer protein.
  • the GLS dimer protein is isoform GAC labeled with ALEXA FLUOR® 488 succinimidyl ester (“488-GAC”).
  • This first labeled GLS dimer protein is a high fluorescence donor protein.
  • a second labeled GLS dimer protein comprising a GLS protein and a fluorescent acceptor attached to the GLS protein is also provided, as illustrated in FIG.
  • this dimer protein is the GLS isoform GAC labeled with the fluorescence acceptor QSY® 9 succinimidyl ester. As illustrated on the right side of the schematic of FIG.
  • binding of the first labeled GLS protein (i.e., 488-GAC) and the second labeled GLS protein (i.e., QSY9-GAC) forms a GLS protein tetramer and results in an interaction between the fluorescent donor (ALEXA FLUOR® 488 succinimidyl ester) and the fluorescence acceptor (QSY® 9 succinimidyl ester) to produce a fluorescence resonance energy transfer at a first level (“FRET” in FIG. 1A ).
  • FRET fluorescence resonance energy transfer at a first level
  • the first labeled GLS dimer protein (i.e., 488-GAC) and the second labeled GLS dimer protein (i.e., QSY9-GAC) are contacted (e.g., brought into contact with each other) under conditions effective for the first labeled GLS dimer protein and the second labeled GLS dimer protein to bind and form a GLS protein tetramer, as illustrated in FIG. 1A .
  • the GLS protein tetramer may then be contacted with a candidate compound.
  • the method further involves detecting whether said contacting with the candidate compound alters the fluorescence resonance energy transfer at the first level.
  • the FRET pair experiences a fluorescence resonance energy transfer at a particular level determined by the donor and acceptor. If, after coming into contact with a candidate compound, the fluorescence resonance energy transfer is unaltered, the candidate compound is determined to neither inhibit nor stabilize GLS protein tetramer formation. If, on the other hand, the fluorescence resonance energy transfer is altered (e.g., is above or below the fluorescence resonance energy transfer of the tetramer in the absence of the candidate compound), then the candidate compound is determined to inhibit or stabilize tetramer formation of GLS protein.
  • this method of the present invention is carried out with a population of dimer proteins comprising donors and a population of dimer proteins comprising acceptors. Under certain conditions, about one-half of the dimers will form tetramers to produce a fluorescence resonance energy transfer (e.g., will establish an equilibrium of dimers:tetramers).
  • the population of dimers:tetramers is contacted with a candidate compound.
  • a candidate compound capable of stabilizing tetramer formation of GLS protein will cause a shift in the equilibrium of dimers:tetramers to increase the number of tetramers formed and, thereby, alter the detectable level of fluorescence energy transfer.
  • a candidate compound capable of inhibiting tetramer formation of GLS protein will cause a shift in the equilibrium of dimers:tetramers in the opposite direction to decrease the number of tetramers formed and, thereby, alter the detectable level of fluorescence energy transfer.
  • FIG. 5A This phenomenon is illustrated in FIG. 5A , in the line representing (+) 10 ⁇ M BPTES.
  • a GLS dimer protein labeled with a donor group emits a fluorescence that is quenched upon coming into contact with a GLS dimer protein labeled with an acceptor group (“Acceptor”).
  • Acceptor an acceptor group
  • This quenching in fluorescence upon contact between the donor and acceptor occurs as the fluorescence of the donor is absorbed by the acceptor.
  • a candidate compound (“968/BPTES”)
  • 968 and BPTES are compounds that stabilize tetramer formation.
  • contact of the FRET pair of GLS dimers by a compound that stabilizes tetramer formation caused additional formation of FRET pairs and, as a result, further absorbance by the acceptor from the donor.
  • kits for compounds that inhibit or stabilize tetramer formation.
  • the kit includes a first labeled GLS dimer protein comprising a GLS protein and a fluorescent donor attached to the GLS dimer protein.
  • a second labeled GLS dimer protein comprising a GLS protein and a fluorescent acceptor attached to the GLS protein. Binding of the first labeled GLS protein and the second labeled GLS protein forms a GLS protein tetramer and results in an interaction between the fluorescent donor and the fluorescent acceptor which produces a fluorescence resonance energy transfer.
  • a mouse kidney type glutaminase isoform 1 (KGA, NP_001074550.1, which is hereby incorporated by reference in its entirety (SEQ ID NO:7)) and isoform 2 (GAC, NP_001106854.1, which is hereby incorporated by reference in its entirety (SEQ ID NO:3)) plasmid (residues 72-603 for GAC, 72-674 for KGA) was cloned into a pET23a vector containing an N-terminal histidine (His)-tag and thrombin cleavage site.
  • His N-terminal histidine
  • the expressed protein was initially purified using Co 2+ affinity beads (Clontech), after which the His-tag was cleaved with human thrombin (Haemetologic Technologies) overnight at 4° C. and subsequently purified by anion exchange (GE healthcare) and gel filtration chromatography.
  • Purified GAC or KGA was stored in a high salt containing buffer (20 mM Tris-HCl pH 8.5, 500 mM NaCl, 1 mM NaN 3 ) and stored at ⁇ 80° C. following snap freezing in liquid N 2 for long term use.
  • the labeling reaction was quenched with 150 mM Tris-HCl pH 8.5 and unreacted probe was separated from labeled-enzyme using a PD10 desalting column eluting labeled-GAC back into the high salt containing buffer.
  • Fluorescence experiments were performed using a Varian Carry Eclipse Fluorometer in the counting mode. Excitation and emission wavelengths were 490 and 520 nm, respectively. Experiments were all prepared as one-mL samples and stirred continuously at 20° C. in 50 mM Tris-Acetate pH 8.5, 0.1 mM ethylenediaminetetraacetic acid (EDTA).
  • EDTA ethylenediaminetetraacetic acid
  • BPTES or 968 (10 ⁇ M or 25 ⁇ M, respectively) was added following equilibration of a sample of 200 nM of QSY® 9-WT GAC and 20 nM 488-WT GAC. Both BPTES and 968 were prepared in DMSO, and appropriate dilutions were made so that less than 2% (v/v) DMSO was added to an experimental sample.
  • F the normalized fluorescence at given drug concentration (i.e., F/F 0 )
  • F sat is the normalized fluorescence at saturating concentrations of drug, as shown in FIG. 7A .
  • NADH fluorescence was measured (340 nm/460 nm excitation/emission, 10 nm/10 nm excitation/emission slits) every minute with 30 second orbital shaking and 30 second rest between each reading for 10 cycles (i.e., 9 minutes).
  • Three wells were prepared for each experimental condition (i.e., each concentration of compound) alongside one well where 2 ⁇ L of DMSO was added in place of inhibitor and one well that contained the small molecule inhibitor but no GAC was added.
  • 488-fluorescence (F) was normalized to the DMSO control (F 0 ) immediately adjacent to the experimental condition.
  • Quenching was quantified by subtracting the normalized fluorescence by one (i.e., 1-F/F 0 ). For compounds that emitted fluorescence within the observed range, fluorescence measured in the well that contained the compound but lacked GAC was used to subtract added fluorescence due to the compound. Similarly, samples were analyzed for NADH fluorescence by subtracting the evolved fluorescence in the experimental condition by the NADH fluorescence evolved in the well that contained the added compound but no GAC. Percent inhibition at each drug concentration was calculated using the adjacent DMSO control.
  • the second step was initiated by the addition of 200 ⁇ L of 12 Units/ ⁇ L GDH, 2 mM NAD + , 100 mM hydrazine (Sigma), and 100 mM Tris-HCl pH 9.2 was on top of the first quenched reaction and incubated 45 minutes at 23° C. before reading NADH absorbance. Glutamate produced by the first reaction was equated to NADH measured from reaction two using the extinction coefficient of NADH (6,220 M ⁇ 1 cm ⁇ 1 ) and a standard curve of a glutamate titration prepared as 25 ⁇ L in step one.
  • BPTES a well characterized inhibitor of GAC
  • the labeling of recombinant GAC was shown to be both rapid and stoichiometric, and did not influence the oligomeric state of the 488-labeled GAC when compared to unlabeled GAC using analytical gel filtration ( FIG. 1B ).
  • the site of covalent modification was shown to be within the conserved glutaminase domain, by means of mass spectrometry identification of peptide fragments produced by partial trypsin digestion and separation by SDS-PAGE ( FIGS. 8A-C ). Indeed, efficient fluorescence quenching was observed when the fluorescence of the 488 labeled GAC was monitored upon addition of the acceptor labeled QSY® 9-GAC ( FIG. 1C ).
  • FIG. 2A highlights critical contacts identified at the GAC tetramer interface ( FIG. 2A , bottom inset), as well as the GAC dimer interface ( FIG. 2A , top inset).
  • MALS multi-angle light scattering downstream of size exclusion chromatography
  • FIG. 6A depicts a model of the enzyme activity assay coupled with the 968-binding assay, where the activity of 488-GAC can be monitored following the interaction of 968 by detecting the NADH fluorescence that results from the coupled glutamate dehydrogenase (GDH) reaction.
  • GDH catalyzes the conversion of glutamate (i.e., the product of GAC activity), to ⁇ -ketoglutarate through the reduction of NAD + (which is non-fluorescent), to NADH (which is highly fluorescent).
  • the assay presented here is a novel tool to monitor both the binding of 968-like molecules, but not BPTES-like molecules, to GAC and the activity of the enzyme in a real-time fluorescent readout using 488 fluorescence (520 nm emission) and NADH fluorescence (460 nm emission) making it highly adaptable to high-throughput screening.
  • FIG. 7B illustrates that by simply decreasing the concentration of GAC from 50 nM to 5 nM, 968 was able to inhibit GAC activity with higher efficacy and potency.
  • 968-mediated inhibition of GAC activity correlates extremely well with its inhibition of oncogenic transformation as read out by inhibition of oncogene-induced foci formation. Taking these observations into consideration, the 488-labeled monomer mutant would provide an efficient means for screening a compound's ability to bind to the 968 allosteric site.
  • Glutamine metabolism is a central metabolic pathway that has been shown to play a vital role in a variety of physiological conditions, ranging from DNA repair in response to ultra-violet radiation (Jeong et al., “SIRT4 has Tumor-Suppressive Activity and Regulates the Cellular Metabolic Response to DNA Damage by Inhibiting Mitochondrial Glutamine Metabolism,” Cancer Cell 23(4):450-463 (2013), which is hereby incorporated by reference in its entirety), glutamate toxicity that often accompanies strokes or HIV infection (Ye et al., “Il-1 ⁇ and TNF-a Induce Neurotoxicity Through Glutamate Production: A Potential Role for Neuronal Glutaminase,” J .

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